Whole-cell FACS was carried out as described in detail in Sabatinos and Forsburg (2009 , 2015 ) and Sabatinos et al. (2015 (link)) with minor changes. To perform cell cycle analysis or microscopy, cells were fixed in 70% ethanol. Following rehydration in 50 mM sodium citrate, cells were treated with 1 µM SytoxGreen (Invitrogen, Carlsbad, CA) plus 10 µg/ml RNase A and incubated at 36°C for 1–2 h. Samples were then sonicated before being analyzed on a FACScan machine (BD Biosciences, San Jose, CA). DAPI staining was done by first rehydrating fixed cells in PBS and placing them to dry on positively charged glass slides. Mount solution (50% glycerol and 1 µg/ml DAPI) was then applied before cells were photographed on a Leica DMR wide-field epifluorescence microscope equipped with a 63×/1.62 NA Plan-Apo objective lens, 100-W Hg arc lamp for excitation, and a 12-bit Hamamatsu ORCA-100 CCD camera. Images were acquired using OpenLab version 3.1.7 (ImproVision, Lexington, MA) software and analyzed with ImageJ.
Orca 100 ccd camera
Manufactured by Hamamatsu Photonics
The Orca 100 CCD camera is a high-performance digital imaging device designed for scientific and industrial applications. It features a cooled CCD sensor that provides low-noise, high-sensitivity image capture. The camera is capable of capturing images with high resolution and dynamic range, making it suitable for a variety of imaging tasks.
Automatically generated - may contain errors
Lab products found in correlation
Dmr wide field epifluorescence microscope, by Hamamatsu Photonics (1 mentions)
Openlab version 3, by PerkinElmer (1 mentions)
Sytox green, by Thermo Fisher Scientific (1 mentions)
Facscan machine, by BD (1 mentions)
80i epifluorescence microscope, by Nikon (1 mentions)
Cy3 conjugated donkey anti rabbit igg, by Jackson ImmunoResearch (1 mentions)
Metamorph 7, by Molecular Devices (1 mentions)
Ixon x3897 camera, by Oxford Instruments (1 mentions)
4 protocols using orca 100 ccd camera
1
Whole-cell FACS for Cell Cycle Analysis
2
Immunofluorescence Staining of Mouse Kidneys
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Fixation of mouse kidneys, preparation of cryostat sections, antigen retrieval, permeabilization and incubation with antibodies were performed as described elsewhere34 (link)47 (link)48 (link). Affinity-purified rabbit polyclonal antibodies were used at the following dilutions: anti-DMXL1 (1:10), anti-DMXL2 (1:20), anti-NCOA7 (1:100), anti-OXR1 (1:50) and anti-WDR7 (1:50). The affinity-purified chicken polyclonal anti-ATP6V1A antibody was used at 1:800. Secondary Cy3-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) and Alexa Fluor® 594-conjugated goat anti-rabbit IgG (Life Technologies/Thermo Fisher Scientific) antibodies were used at 1:800, an FITC-conjugated donkey anti-chicken IgY antibody (Jackson ImmunoResearch) at 1:60 and an Alexa Fluor® 488-conjugated donkey anti-chicken IgY antibody (Jackson ImmunoResearch) at 1:200. Images were obtained using a Nikon 80i epifluorescence microscope (Nikon Instruments, Melville, NY) equipped with an Orca 100 CCD camera (Hamamatsu, Bridgewater, NJ).
3
Imaging Adult Worms with BDM
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Images were taken using an Olympus PlanAPO with a 100 × 1.4 NA objective and an ORCA100 CCD camera (Hamamatsu). Young adult worms were immobilized with 30 mg/ml BDM (2,3-Butanedione monoxide, Sigma). Image stacks were captured and the maximum intensity projections were obtained using Metamorph 7.1 software (Molecular Devices).
4
High-Resolution Microscopy Techniques for Cellular Imaging
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Cells and tissues were visualized using a Leica microscope (model DMR, Melville NY) fitted with 40X (PL Fluotar, NA 1.0) and 63X (PL APO, NA 1.32) objectives. Images on the DMR microscope were acquired using an Orca 100 CCD camera (model C4742-95; Hamamatsu, Bridgewater, NJ) and analyzed using ImageJ software (NIH version 2.0.0) or Metamorph. For N-SIM analysis, the samples were illuminated with spatially high-frequency patterned excitation light (100X objective lens, NA 1.49; TiE N-SIM microscope [Nikon] and iXON X3 897 camera [Andor Technology]). Images were reconstructed and analytically processed to reconstruct subresolution structure of the samples using Elements version 4 software (Nikon). For siNEDD8 and siCOPS3 EGFR internalization, the Axioimager and the Apotome2 was used with the 40x objective and a MIP of the Z sections was used.
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